水包油佐剂对小鼠骨髓来源树突状细胞蛋白表达的影响  

Effect of oil-in-water adjuvant on protein expression of dendritic cells derived from mouse bone marrow

作　　者：年悬悬 施金荣 龚铮 刘京 李军英 张家友 杨晓明 Nian Xuan-xuan;Shi Jin-rong;Gong Zheng;Liu Jing;LI Jun-ying;Zhang Jia-you;Yang Xiao-ming(2th Research Department of Viral Vaccine,Wuhan Institute of Biological Products Co.,Ltd.,National Engineering Technology Research Center for Combined Vaccines,Wuhan 430207,Hubei Province,China)

机构地区：[1]武汉生物制品研究所有限责任公司病毒性疫苗研究二室,湖北武汉430207 [2]国家联合疫苗工程技术研究中心,湖北武汉430207

出　　处：《微生物学免疫学进展》2022年第1期8-14,共7页Progress In Microbiology and Immunology

基　　金：四价流感病毒裂解疫苗的研制(2016ZX09106003-008)。

摘　　要：目的探索未成熟的小鼠骨髓来源树突状细胞(bone marrow derived dendritic cells,BMDCs)的分离诱导培养方法,并研究水包油佐剂对BMDCs蛋白表达的影响。方法解剖小鼠获得小鼠骨髓细胞,分析在骨髓细胞诱导培养获得未成熟的BMDCs的过程中,红细胞裂解液(red blood cell lysis buffer,RBC)、诱导剂IL-4、rmGM-CSF及培养容器(6孔板或细菌培养皿)对获得未成熟BMDCs的纯度和成熟度的影响。用水包油佐剂AddaVax刺激培养收获的BMDCs 24 h后,检测BMDCs表面分子的表达差异,以及通过蛋白组学方法检测BMDCs细胞所有蛋白的表达差异。结果一只小鼠的骨髓细胞诱导培养6 d,培养条件为无RBC处理、细菌培养皿培养,获得的BMDCs细胞数最高,为1.2×10^(7)/只,且BMDCs细胞的纯度较高(约60%),与在6孔板中IL-2和rmGM-CSF共同诱导获得的BMDCs纯度差异无统计学意义(P>0.05)。各种培养方法诱导BMDCs表面成熟标志CD86、CD40的表达分别<40%、<70%。水包油佐剂AddaVax诱导BMDCs表面分子MHCⅡ、CD40、CD80和CD86的表达均>80%。蛋白组学分析显示,AddaVax促进了BMDCs生物调节以及免疫应答相关蛋白NRF2、KEAP1、P16、P53、Rb、BGFR、Ras、Raf、TRADD、ARIDIA、BAF155、ARID2、SWI/SNF的表达。结论分离的骨髓细胞不需要进行RBC处理,在细菌培养皿用rmGM-CSF诱导培养骨髓细胞6 d即可制备大量未成熟的BMDCs。水包油佐剂AddaVax促进了未成熟BMDCs的增殖、分化、成熟,并且促进了BMDCs免疫相关通路蛋白的表达。Objective To evaluate the isolation and culture method for immature murine bone marrow-derived dendritic cells(BMDCs),and explore the effect of oil-in-water adjuvants on the protein expression of BMDCs.Methods Bone marrow cells treated or untreated with Red Blood Cell Lysis Buffer(RBC)were cultured in 6-well plates or bacterial culture dishes,and stimulated by GM-CSF alone or with IL-4,and then the purity and maturity of obtained BMDCs was analyzed.After 24 hours of the oil-in-water adjuvant AddaVax stimulation,the surface markers and other proteins expression of BMDCs were detected by proteomics analysis.Results The bone marrow cells harvested from a mouse were induced and cultured for 6 days.The number of BMDCs cultured in bacterial culture dish without RBC treatment was 1.2×10^(7),and the purity was about 60%,which was higher than that cultured in 6-well plate.The expression of CD86 and CD40 on the surface of BMDCs induced with various culture methods was lower than 40%and 70%,respectively.The expression of MHCⅡ,CD40,CD80 and CD86 on the surface of BMDCs induced by AddaVax exceeded 80%.Proteomics analysis showed that AddaVax promoted biological regulation and the expression of immune response-related proteins NRF2,KEAP1,P16,P53,Rb,BGFR,Ras,Raf,TRADD,ARIDIA,BAF155,ARID2,SWI/SNF.Conclusion A large number of immature BMDCs can be prepared by culturing bone marrow cells with rmGM-CSF in a bacterial culture dish for 6 days without RBC treatment.AddaVax promotes the proliferation,differentiation and maturation of immature BMDCs,and the proteins expression in immune-related pathways.

关 键 词：骨髓 树突状细胞 水包油佐剂 蛋白组学 信号通路 

分 类 号：R392[医药卫生—免疫学]